Developing effective antiviral drugs requires a detailed understanding of the molecular mechanisms underlying the targeted host-pathogen interaction. Specifically, precise structural models of these interactions can provide mechanistic details with atomic resolution to assist in the efficient development of novel compounds against pathogens of significant health relevance. The influenza virus is a prime example of one such pathogen. Novel strains of the influenza virus develop annually via infection and replication in a number of animal hosts, including humans. The relative ability of these strains to cause disease, or virulence, is determined by a number of interactions between viral and cellular proteins. Although influenza non-structural protein 1 (NS1) is known to play a critical role in virulence, there is a fundamental gap in our knowledge of the genetic and structural determinants that facilitate the multiple strain-dependent functions attributed to NS1 in the host cell. It is therefore our long-term goal to understand the molecular mechanisms that underlie strain-dependent function of NS1. The objective of this application is to structurally characterize interactions with two cellular proteins (CPSF30 and RIG-I) that are important for the activation of the innate immune response. Our central hypothesis is that structural and dynamic features unique to certain NS1 variants account for the diverse array of functions attributed to NS1. The rationale that underlies the proposed research is that elucidating structure- function relationships between NS1 and its cellular interaction partners will aid in the development of antiviral drugs that target these critical interactions known to modulate virulence. Our central hypothesis will be tested by pursuing three specific aims: 1) structurally and functionally characterize the multiple interactions between NS1 and RIG-I, 2) determine the role of microsecond-millisecond (s-ms) motions in proper function of the NS1 effector domain (NS1ED), and 3) determine the mechanism of action by which JJ3297 suppresses influenza replication. In Aim 1, NMR spectroscopy and mutant recombinant influenza viruses will be used to structurally and functionally characterize the multiple interactions between NS1 and RIG-I. In Aim 2, relaxation dispersion experiments will be used to determine the role that protein dynamics play in facilitating the interaction between the NS1ED and CPSF30 and intracellular localization of NS1. In Aim 3, NMR spectroscopy will be used to determine the mechanism of action by which JJ3297 suppresses influenza replication. Our innovative approach will be the first investigation into how protein dynamics and strain specific structural variations facilitate proper function of NS1 in the context of viral replication and pathogenicity. This will also be the first systematic study to determine functional variations in NS1 between multiple strains of influenza. The proposed research is significant because it will define the molecular mechanisms underlying NS1 functions shown to modulate influenza virulence. By defining these molecular mechanisms, this proposal will inform efforts in developing influenza antiviral drugs targeting NS1, thereby supporting the overall mission of the NIH.